The present invention relates to a novel tumor suppressor gene, referred to herein as SSeCKS, its encoded protein, and methods of use thereof. It is based, at least in part, on the discovery of a SSeCKS gene which encodes a substrate of protein kinase C that functions as both a mitogenic regulator as well as a tumor suppressor.
The inactivation of several tumor suppressor gene families (for example, those encoding p53, Rb, and APC) as a result of mutation is acknowledged to contribute to oncogenicity of several types of human cancers (Levine, 1993, Ann. Rev. Biochem. 62:623-651). Many of these so-called class I tumor suppressor genes (Lee et al., 1991, Proc. Natl. Acad. Sci. U.S.A. 88:2825-2829) were identified and isolated following cumbersome pedigree and cytogenetic analyses (Sager, 1989, Science 246:1406-1412). Recently, another class of genes (class II) whose expression is known to be down-regulated in tumor cells has been shown by gene transfer techniques to encode potential tumor suppressors. These include nonmuscle xcex1-actinin, tropomyosin I, CLP, retinoic acid receptor xcex21, and interferon regulatory factor (Gluck et al., 1993, Proc. Natl. Acad Sci. U.S.A. 90:383-387; Hirada et al., 1993, Science 259:971-974; Hogel et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:985-989; Mishra et al., 1994, J. Cell. Biochem. 18(Supp. C):171; Plasad et al., 1993, Proc. Natl. Acad. Sci. U.S.A. 90:7039-7043). Additional tumor suppressor gene families such as the maspin gene, rrg, and NO3 (Contente et al., 1993, Science 249:796-798; Ozaki et al., 1994, Cancer Res. 54:646-648; Zou et al., 1994, Science 263:526-529) were isolated by subtractive hybridization techniques designed to identify down-regulated genes. The ability of these genes to reverse an array of oncogenic phenotypes following gene transfer and over-expression supports the possibility for novel therapeutic modalities for cancer.
The present invention relates to a novel tumor suppressor gene, SSeCKS. It is based, at least in part, on the discovery of a gene, hitherto referred to as xe2x80x9c322xe2x80x9d (Lin et al., 1995, Mol. Cell. Biol. 15:2754-2762) but now referred to as SSeCKS, which was found to be down-regulated in certain transformed cells. Further, the SSeCKS gene product has been found to be a substrate of protein kinase C, and has been shown to act as a mitogenic regulator and as an inhibitor of the transformed phenotype.
In various embodiments, the present invention relates to the SSeCKS gene and protein, and in particular, to rat and human SSeCKS gene and protein. Furthermore, the present invention provides for the use of such genes and proteins in diagnostic and therapeutic methods.
FIG. 1. Northern blot analysis of SSeCKS RNA levels in NIH 3T3 cells versus NIH/v-src transformed cells.
FIG. 2. Southern blot analysis showing that the decreased level of SSeCKS RNA in NIH/v-src cells is not due to gross deletion or translocation of the SSeCKS allele (A), and restriction map of SSeCKS (B).
FIG. 3. Nucleic acid SEQ ID NO: 1 (top line, lower case letters) and deduced amino acid SEQ ID NO: 2 (lower line, capital letters) sequence of rat SSeCKS cDNA encoding an active truncated form of SSeCKS.
FIG. 4. Northern blot analysis showing that the transcription of SSeCKS is suppressed relatively soon after the activation of a ts-src allele (A) or the addition of fetal calf serum (FCS) to starved rodent fibroblasts (B).
FIG. 5. Northern blot analyses showing levels of SSeCKS transcripts in oncogene-transformed Rat-6 fibroblasts.
FIG. 6. Results of in vitro transcription-translation of SSeCKS cDNA.
FIG. 7. Proliferation of cells overexpressing SSeCKS (A and B).
FIG. 8. xe2x80x9cZooxe2x80x9d Southern blot of SSeCKS probe to genomic DNA from various species.
FIG. 9. Northern blot analysis showing tissue-specific expression of SSeCKS in mice.
FIG. 10. Schematic diagram of SSeCKS protein.
FIG. 11A-I. Nucleic acid SEQ IN NO: 3 sequence of rat cDNA encoding full-length SSeCKS and deduced amino acid sequence.
FIG. 12. In vitro transcription and translation of SSeCKS. One xcexcg of plasmid DNA encoding the full-length SSeCKS cDNA or a N-terminally truncated SSeCKS cDNA (clone 13.2.2) were incubated in a coupled T7 transcription/translation reaction (TNT; Promega) containing [35S]-methionine as described in section 7.1. One tenth of the labeled products were analyzed by SDS-PAGE followed by fluorography. Protein size markers are shown at left. Note that a shortened version of SSeCKS, synthesized from an internal ATG start site in clone 13.2.2, is not produced in the context of the upstream ATG start site in the full-length SSeCKS cDNA in in vitro reactions.
FIGS. 13A-C. Glutathione S-transferase fusion constructs of SSeCKS domains. Secondary structural analysis of SSeCKS predicted a rod-like molecule with a high degree of hydrophilicity and amphipathic helices, and a concentration of Chou-Fasman turns (Chou and Fasman, 1978, Advances in Enzymology 47:45-147) from residues 400-900 (13B and 13C). The turns in this region were not recognized by the Robson-Garnier algorithm (Garnier et al., 1978, J. Mol. Biol. 120:97-120), as shown in 13C. Four concentrations of predicted PKC phosphorylation sites (B/TXK/R or K/RXXS/T) were also identified (13A, white boxes; numbered 1-4). The black bars (13A) indicate the sizes and names of GST-SSeCKS fusion constructs containing individual or combinations of the predicted PKC sites.
FIGS. 14A-B. In vivo phosphorylation of SSeCKS by PKC. Confluent Rat-6 cells grown overnight in DEM lacking calf serum were starved of phosphate for 2 hours and then labeled for 4 hours with [32 P]orthophosphate. At the end of the labeling period, some cells were treated with 200 nM PMA (lane b, 2 min; lanes c and d, 15 min) and the PKC-specific inhibitor, bis-indolylmaleimide (lane d, 30 min). SSeCKS protein was immunoprecipitated from equal aliquots (400 xcexcg) of lysates from untreated (lane a) or treated cells (lanes b-d), and western blotted onto a PVDF membrane (14A). 14B represents immunoblotting using rabbit anti-SSeCKS serum (showing equal amounts of SSeCKS protein loaded) whereas the upper panel represents autoradiography of the blotted protein (showing an increase in 32PO4-labeling of SSeCKS following PMA treatment). The 280/290 kDa doublet (unresolved in this gel) is indicated by an arrow, and the minor 240 kDa form of SSeCKS can be detected in the upper panel. A better resolution of these SSeCKS species is shown in FIG. 22.
FIGS. 15A-B. In vitro phosphorylation of SSeCKS by PKC. GST and GST/322 fusion protein (see FIG. 13) were expressed and purified from bacteria as described in section 7.1 (15A). Five xcexcg of the GST samples were added to PKC assays containing [32P]-xcex3-ATP in the presence or absence of the PKC peptide inhibitor (19-36). The products were then bound to glutathione-Sepharose beads, precipitated and washed, and analyzed by SDS-PAGE and autoradiography (15B). Protein size markers are indicated on the appropriate sides. Radioactive labeling was detected in GST-322 (160 kDa) only.
FIGS. 16A-B. Phospholipid preference for the in vitro phosphorylation of SSeCKS by PKC. Myelin basic protein, MBP (16A), GST-322 and GST proteins (16B) were phosphorylated in vitro as in FIG. 15, in the presence or absence of various lipids including phosphatidylserine (PS), phosphatidylcholine (PC) or phosphatidylinositol (PI). In some cases, excess PKC peptide inhibitor (19-36) was added as in FIG. 15. The extent of labeling in the peptide substrates was determined by spotting the reaction products on phosphocellulose discs (Whatman), precipitating peptides with washes of 5% trichloroacetic acid, followed by scintillation counting.
FIGS. 17A-B. Co-precipitation of SSeCKS with PKC. 17A: GST-1322 fusion protein (see FIG. 13) was expressed and purified from bacteria as described in section 7.1. 17B: RIPA lysates (1 mg protein per sample) from Rat-6 or Rat-6/PKC-xcex1 overexpressor cells, or purified rabbit brain PKC (20 ng; xe2x80x9cP-PKC-xcex1xe2x80x9d) were incubated for 4 h at 4xc2x0 C. with fifty xcexcg of GST-1322 (or GST alone) in the presence or absence of 0.37 mg/ml phosphatidylserine (PS). The proteins were then precipitated with glutathione-Sepharose beads, washed and western blotted as described in section 7.1. The filters were probed with MAB specific for PKC type III (UBI). The lane to the right is loaded with 20 ng of purified rabbit brain PKC-xcex1.
FIGS. 18A-I. In vitro phosphorylation of PKC sites 1-4 on SSeCKS. 18A and 18B: Five xcexcg of various GST-SSeCKS fusion proteins containing individual or combinations of the predicted PKC phosphorylation sites (1-4) in SSeCKS, were subjected to an in vitro PKC assay containing [32P]-xcex3-ATP and analyzed as in FIG. 15. 18C-F and 18G-I: Expression and purification of the GST-SSeCKS fusion proteins. Fifty xcexcg aliquots of bacterial lysate from uninduced (lane a) or induced (lane b) bacteria, or 5 xcexcg of GST-SSeCKS fusion protein eluted from glutathione-Sepharose columns (lane c), were analyzed by SDS-PAGE, and then stained with coomassie blue. Arrows indicate the size of the unfragmented protein product.
FIG. 19. SSeCKS is resistant to heat denaturation. 150 xcexcg aliquots of Rat-6 cell lysate were boiled for 5 min in the absence of SDS and then debris removed by low speed centrifugation (1 K rpm at 4xc2x0 C.). Lane c represents supernatant which was applied directly onto an SDS/poly-acrylamide (5%) gel, whereas lane b was boiled supernatant first immunoprecipitated with rabbit anti-SSeCKS serum (lane b) as described in section 7.1 (IgH is immunoglobulin heavy chain recognized by the AP-labeled sheep anti-rabbit Ig secondary antibody). Lane a contains the SSeCKS protein remaining in the lysate after the immunoprecipitation in lane b. Note that under these conditions,  greater than 95% of the SSeCKS protein is usually immunoprecipitated. Lane d contains 150 xcexcg of unboiled lysate run directly on the gel.
FIG. 20. SSeCKS expression in src- and ras-transformed cells. 250 xcexcg of total protein from Rat-6, Rat-6/src and Rat-6/ras (1) cell lysates was analyzed by immunoblotting for SSeCKS as described in section 7.1. In addition to the 240 kDa (larger arrow) and 280/290 kDaa (small arrow) forms of SSeCKS found in untransformed cells, a 305 kDa form was detected in Rat-6/ras cells and to a lesser extent in Rat-6/src cells. The relative level of SSeCKS in src- and ras-transformed cells compared with Rat-6 cells (as defined by densitometry) is 15- and 8-fold lower, respectively.
FIGS. 21A-J. Immunofluorescence analysis of SSeCKS cellular localization. Subconfluent (G-J) or confluent (A-F) cultures of 3Y1 rat fibroblasts were fixed and analyzed for SSeCKS (A, C, E, H-J) or actin (B, D, F) expression as described in section 7.1. Panel G represents cells incubated with pre-immune rabbit sera. SSeCKS was present throughout the cytoplasm in subconfluent and confluent cells (e.g.-panel J, xe2x80x9ccyxe2x80x9d; panel A) and in the paranucleus (e.g.-panel J, xe2x80x9cpnxe2x80x9d). SSeCKS was also enriched in focal contact sites (e.g.-panel H, arrows), in podosomes (e.g.-panel I, xe2x80x9cpxe2x80x9d) and at the cell edge (panel J, xe2x80x9ccexe2x80x9d; panel A). Confluent 3Y1 cells showed mostly cytoplasmic staining of SSeCKS (A), possibly associated with cortical actin but not with actin stress fibers (B). After 10 min treatment with 200 nM PMA, SSeCKS moved away from plasma membrane sites towards the paranucleus (C), simultaneous with the ruffling of actin fibers at the membrane (D). The inward movement of SSeCKS and the ruffling of actin became more pronounced after 60 min treatment with PMA (E and F, respectively).
FIG. 22. SSeCKS does not enter a soluble cellular component after short-term PMA treatment. Confluent Rat-6 or Rat-6/PKC-xcex1 overexpressor cell cultures were grown overnight in DEM lacking calf serum, and then treated with PMA (1.6 xcexcM) for 30 min or mock-treated for the same duration. The cells were lysed and spun at low speed (1.5 K rpm), yielding a pellet (P1) and supernatant (S1). The S1 component was fractionated by differential centrifugation into plasma membrane (P100) and soluble (S100subcellular components as described in section 7.1. 50 xcexcg aliquots of P1 and P100, and 25 xcexcg aliquots of S100 were then immuno-blotted using rabbit anti-SSeCKS as in FIG. 14. The SSeCKS isomers (240, 280, and 290 kDa) are shown in relation to a 220 kDa marker protein (myosin heavy chain).
FIG. 23A. Identification of a human SSeCKS gene homologue. 2 xcexcg of poly A+mRNA from various tissues was probed with radiolabeled rat SSeCKS cDNA under conditions of stringent hybridization. The tissue distribution and message size in humans is similar to that in mice (FIG. 9).
FIG. 23B. Western blot of SSeCKS expression in various mouse tissues and in human fibroblasts (WI-38) using antibody directed against rat SSeCKS protein. Note that anti-SSeCKS sera recognizes a 280/290 kDa doublet in human cells. Taken with the data in FIG. 23A, this indicates that humans encode an SSeCKS homologue.
FIG. 24. Northern blot of RNA prepared from various human tumor cell lines, using radiolabeled rat SSeCKS cDNA as a probe.
FIG. 25A and B. Full-length SSeCKS decreases v-src-induced colony formation in soft agar.
FIG. 26. Amino acid sequences associated with myristylation and palmitylation (SEQ ID NO: 5 to 10.
FIG. 27. Inhibition of proliferation of cells in tetracycline-containing (+) and tetracycline-free (xe2x88x92) media by SSeCKS, encoded by a tetracycline-repressed construct and expressed in the absence of tetracycline.
FIG. 28. Polyacrylamide gel electrophoresis (PAGE) showing labeling of SSeCKS, encoded by a tetracycline-repressed construct and expressed in the absence of tetracycline, with tritiated myristate.
FIG. 29. Northern blot showing expression of SSeCKS RNA in the testes of normal Swiss mice and weaver mutant mice.
FIG. 30A-D. Photomicrographs showing S24 cells transfected with tetracycline-repressed SSeCKS construct in tetracycline-free (30A and 30B) and tetracycline-containing medium (30C and 30D).
FIG. 31A-D. Photomicrographs of tetracycline-repressed SSeCKS transfected S24 cells stained with fluorescent labeled antibodies to SSeCKS (31A and 31C) and F-actin (31B and 31D) in the presence (31A and 31B) and absence (31C and 31D) of tetracycline.
FIG. 32A-H. Overexpression of SSeCKS delays the formation of vinculin-associated adhesion plaques (ap). S24 cells (see FIG. 1) grown in the absence of tetracycline for 1(a,b,g,h), 3(c,d) and 4(e,f) days were stained for SSeCKS (a,c,e and g) and vinculin (b,d,f and h). After 1 day, adhesion plaques were detected only in the cell not over-expressing SSeCKS (left cell, panel a/b). After 3 days, adhesion plaques began to form in the SSeCKS overexpressor cells but were not located at the cells"" leading edges (le). After 4 days, adhesion plaques were detected at the leading edge in the SSeCKS overexpressor cells. Panels g and h show the inverse expression pattern of SSeCKS and vinculin in filopodia of overexpressor (S24) and non-overexpressor cells (n).
FIG. 33A-H. Photomicrographs depicting fluorescent staining with anti-actin antibodies (33B, 33D, 33F, and 33H) or anti-SSeCKS antibodies (33A, 33C, 33E, and 33G) in cell-wounding experiments.